Cell culture method, cell culture device, method for counting subsject matters to be counted in container and device for counting

ABSTRACT

Cell aggregate formation control and cell aggregate disintegration control are carried out with respect to cells in a culture container to increase cell proliferation efficiency. The number of the cells in the culture container is counted without disassembly of a culture system and irrespective of the density of the cells. 
     An external force is applied to the culture container to carry out at least one of cell aggregate formation control and cell aggregate disintegration control with respect to cells in the culture container, thus culturing the cells. The external force is applied to the culture container by pressing an agitating member onto a top surface of the culture container placed in flat orientation to a predetermined pressing degree, and moving the agitating member in a horizontal direction at a predetermined speed, thus controlling at least one of cell aggregate formation and cell aggregate disintegration with respect to the cells in the culture container. A method for counting counted targets disposed in a liquid enclosed in a container includes adjusting a thickness of at least a part of the container. At least a part of the adjusted part is set as a measurement target region. A number of counted targets in the measurement target region is counted.

TECHNICAL FIELD

The present invention relates to a method for culturing cells and a cellculturing apparatus that are for culturing cells such as cells, tissues,and microorganisms, and to a counting method and a counting apparatus tocount counted targets in a container.

BACKGROUND ART

In recent years, demands exist for efficient large-scale cell culturing,tissues, and microorganisms under artificial environments, in fieldssuch as pharmaceutical manufacturing, gene therapy, regenerative medicaltherapy, and immunotherapy.

In such large-scale cell culturing, especially regarding culturing offloating cells, agitation culturing is commonly practiced using culturevessels equipped with agitating blades. However, agitating blades arenot used with cells subject to damage from external force or cells thatproliferate while forming aggregates. Instead, a widely employed methodis to enclose cells in a culture container where the cells are culturedwhile being kept stationary (with the cells down at the bottom). Then,in accordance with the degree of proliferation of the cells, the methodinvolves transfer to another culture container with a larger bottom areaor increasing the number of containers. Unfortunately, the stationaryculturing is problematic in that as the aggregates of cells grow largerwith the proliferation of cells, this causes a gradual deficiency ofoxygen and nutrients to be fed to the cells, leading to degradedproliferation efficiency.

Additionally, although the transfer to another container involvesagitation of a culture solution to cancel the unevenness of oxygen andnutrients, another problem arises in that the handling during thetransfer causes damage to the cells, leading to degraded proliferationefficiency.

Meanwhile, shaking culture is also widely practiced to constantlyagitate the culture container.

For example, patent document 1 describes a cell culturing apparatus thatuses various patterns, such as rotation and shaking, to move a base onwhich a culture container is placed, thereby agitating a culturesolution in the culture container.

Patent document 2 and patent document 3 describe cell culturingapparatuses that shake a liquid culture medium in a culture containerwhile ensuring that no air bubbles occur, and that supply oxygen bymotion of waves while ensuring that the cells are not damaged.

With the method of shaking by the cell culturing apparatuses, the entireculture medium is intensely agitated. This causes the cells to beseparated from each other and the oxygen and nutrients to be dispersedin the whole area, so that the oxygen and nutrients are suppliedsufficiently to each cell.

Additionally, such cell culturing requires the density of cells in theculture solution to be maintained within an appropriate range inaccordance with the proliferation of the cells.

That is, an excessively high density of cells in the culture solutionprevents a sufficient supply of oxygen and nutrients to each cell,leading to degraded cell proliferation efficiency. Likewise, anexcessively low density of cells in the culture solution preventssufficient securement of cell proliferation efficiency.

In view of this, cell culturing requires a grasp of the density of cellsduring culturing, by appropriately counting the number of cells in aculture solution in a culture container.

For example, patent document 4 discloses a cell culturing apparatus thatproperly maintains the density of cells in a culture solution inaccordance with the proliferation of cells.

In order to measure the number of cells with such cell culturingapparatus, it is conventional practice to: sample a culture solutioncontaining cultured cells through a sampling port coupled to theinterior of the culture container; add a predetermined buffer solutionto the sampled culture solution so as to adjust the density of the cellsin the sampled culture solution to an appropriate density formeasurement; and inject the resulting culture solution into a counterboard that indicates the number of the cells. The number of the cells isread by an operator or a machine to calculate the density of the cells.

Patent document 5 discloses a culture apparatus equipped withphotographing means. According to this culture apparatus, cell imagesare captured periodically and stored.

RELATED ART DOCUMENTS Patent Documents

-   [Patent document 1] USP5057429.-   [Patent document 2] WO2000/66706.-   [Patent document 3] Japanese Translation of PCT International    Application Publication No. JP2007-511231.-   [Patent document 4] WO2008/136371.-   [Patent document 5] WO2007/052718.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

Unfortunately, some kinds of cells have difficulty in cell proliferationif they are separated into individual cells. These kinds of cellsimprove their proliferation efficiency when they adhere in the form ofaggregates of appropriate sizes. Specific examples include adhesivecells such as neural stem cells, embryonic stem cells, hepatic cells,cornea stem cells, pancreatic islet cells, and floating cells such asleukocyte cells.

With these cells, since the conventional stationary culturing involvesintense agitation of the culture container at the time of everytransfer, a problem arises in that the cells are separated from eachother at the time of every agitation, leading to degraded cellproliferation efficiency.

Likewise, the shaking culture with the cell culturing apparatusesdescribed in patent documents 1 to 3 involves intense agitation of theentire culture medium, turning the cells into an individually floatingstate in the culture medium. This makes the cells difficult to form intoaggregates of appropriate sizes, to the detriment of the optimization ofcell proliferation efficiency.

Additionally, counting the number of cells in a culture solution withthe cell culturing apparatus described in patent document 4 requiressampling of the culture solution from the interior of the culturecontainer. This involves disassembly of a culture system at the risk ofcontamination.

Additionally, the culture apparatus described in patent document 5,though capable of acquiring cell images, finds it difficult to measurethe number of cells accurately based on the images and to obtain thedensity of the cells.

Specifically, when the cells in the culture container are photographedby the photographing means described in patent document 5, the number ofthe cells in the cell image is measured, and the number is divided bythe volume of a part of the culture solution defined within the viewfield of the photographing means. Thus, the density of the cells iscalculated.

However, with such direct observation of the cells in the cellcontainer, when the density of the cells is so large that the cellsoverlap with each other as shown in FIG. 24, the number of the cellscannot be measured accurately. Meanwhile, when the density of the cellsis excessively small, it is difficult to prospect the entire density,creating a problem of poor accuracy of the calculated cell density.

A comparison of the use of the photographing means in directly measuringthe cells in a culture container with the use of the conventionalcounter board in actual measurement shows that the thickness of thecounter board is usually approximately 0.1 mm, whereas the thickness ofthe culture container is approximately 1 to 2 cm, which is 100 to 200times the thickness of the conventional counter board.

This means that the number of cells observed through the photographingmeans is 100 to 200 times larger than the number of cells actuallymeasured by the counter board, when the respective volume densities arethe same. Accordingly, the cells in the culture container oftentimesoverlap with each other, and it is difficult to measure the number ofcells by directly observing the cells in the culture container.

In view of this, the inventors have conducted an extensive study, and asa result, successfully obtained a cell density close to an actuallymeasured value by: adjusting the thickness of a culture container to setthe number of cells observed through the photographing means at ameasurable number; and then measuring the number of the cells in theculture container by direct observation.

The present invention has been made in view of the above-describedcircumstances, and it is an object of the present invention to provide:a cell culturing apparatus that carries out formation control anddisintegration control of an aggregate of cells in a culture containerso as to adjust the aggregate to an appropriate size and thereby toimprove cell proliferation efficiency; and a method for culturing cells.

It is another object of the present invention to provide: a countingmethod for a counting target in a container, by which the number ofcells is measured in any density range in a culturing environmentwithout disassembly of the culture system and irrespective of thedensity of proliferated cells; and a counting apparatus.

Means of Solving the Problems

In order to achieve the above-described objects, according to one aspectof the present invention, a method for culturing cells using a culturecontainer includes applying an external force to the culture containerto carry out at least one of cell aggregate formation control and cellaggregate disintegration control with respect to cells in the culturecontainer.

According to another aspect of the present invention, a cell culturingapparatus is to culture cells using a culture container. The cellculturing apparatus includes a loading base on which the culturecontainer is placed, and an agitating member configured to press theculture container to a predetermined pressing degree and movable at apredetermined speed in a horizontal direction. The agitating member isconfigured to move to apply an external force to the culture containerso as to control at least one of cell aggregate formation and cellaggregate disintegration with respect to the cells in the culturecontainer.

According to another aspect of the present invention, a method forcounting counted targets disposed in a liquid enclosed in a containerincludes adjusting a thickness of at least a part of the container. Atleast a part of the adjusted part is set as a measurement target region.A number of counted targets in the measurement target region is counted.

In the method for counting counted targets disposed in a liquid enclosedin a container, the liquid in the container is agitated to equalize thecounted targets in the liquid prior to adjusting the thickness of atleast a part of the container.

According to the other aspect of the present invention, a countingapparatus is to count counted targets disposed in a liquid enclosed in acontainer. The counting apparatus includes a loading base on which thecontainer is placed, and an adjusting member configured to adjust atleast a part of the container including a measurement target region to apredetermined thickness.

The counting apparatus may further include an agitating memberconfigured to agitate the liquid in the container before the adjustingmember adjusts the thickness of the container.

The counting apparatus may further include photographing means, countingmeans, and a driving device. The photographing means is forphotographing counted targets disposed in the container. The countingmeans is for counting a number of the counted targets in a photographedimage. The driving device is configured to, when the number of thecounted targets is outside a predetermined range following the countingby the counting means, drive the adjusting member to adjust at least apart of the container to a predetermined thickness so as to render thenumber of the counted targets in the image within the predeterminedrange.

ADVANTAGEOUS EFFECTS OF THE INVENTION

With the embodiments of the present invention, aggregates are adjustedto appropriate sizes during cell culturing of cells, tissues, andmicroorganisms, thereby improving cell proliferation efficiency.

Also with the embodiments of the present invention, the number of cellsis counted without disassembly of a culture system and irrespective ofthe density of proliferated cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a cell culturing apparatus according toa first embodiment of the present invention.

FIG. 2 is a diagram illustrating a driving device of the cell culturingapparatus according to the first embodiment of the present invention.

FIG. 3 is a schematic side view of the cell culturing apparatusaccording to the first embodiment of the present invention.

FIG. 4 is a diagram illustrating formation of an aggregate of cells anddisintegration of the aggregate of the cells in the present invention.

FIG. 5 is a diagram illustrating a cell culturing apparatus according toa second embodiment of the present invention.

FIG. 6 is a diagram illustrating a cell culturing apparatus according toa third embodiment of the present invention.

FIG. 7 is a diagram illustrating the principle of a counting method ofthe present invention for counted targets in a container.

FIG. 8 is a diagram illustrating a counting apparatus according to afourth embodiment of the present invention.

FIG. 9 is a diagram illustrating a method of adjusting the thickness ofa container (the case of reducing the thickness) using the countingapparatus according to the fourth embodiment of the present invention.

FIG. 10 is a diagram illustrating a method of adjusting the thickness ofa container (the case of increasing the thickness) using the countingapparatus according to the fourth embodiment of the present invention.

FIG. 11 is a diagram illustrating a counting apparatus according to afifth embodiment of the present invention.

FIG. 12 is a diagram illustrating basic positions associated with acounting apparatus according to a sixth embodiment of the presentinvention.

FIG. 13 is a diagram illustrating a state of agitation by the countingapparatus according to the sixth embodiment of the present invention.

FIG. 14 is a diagram illustrating a state of thickness regulation andprecipitation waiting (the case of reducing the thickness) on thecounting apparatus according to the sixth embodiment of the presentinvention.

FIG. 15 is a diagram illustrating a state of microscope observation onthe counting apparatus according to the sixth embodiment of the presentinvention.

FIG. 16 is a diagram illustrating a state of thickness regulation andprecipitation waiting (the case of increasing the thickness) on thecounting apparatus according to the sixth embodiment of the presentinvention.

FIG. 17 is a diagram illustrating kinds of conditions of agitation bythe cell culturing apparatus according to the first embodiment of thepresent invention.

FIG. 18 is a diagram showing cell states as a result of agitation by thecell culturing apparatus according to the first embodiment of thepresent invention under the respective agitation conditions.

FIG. 19 is a diagram illustrating a culture container used in examples 1to 5 of the present invention and comparative example 1.

FIG. 20 shows images of cells of example 1 of the present invention andof comparative example 1.

FIG. 21 is a diagram showing results in example 1 of the presentinvention and comparative example 1.

FIG. 22 is a diagram showing images of cells in examples 2 to 5 of thepresent invention.

FIG. 23 is a diagram showing results in examples 2 to 5 of the presentinvention.

FIG. 24 is a diagram showing a conventional method for counting countedtargets disposed in a container.

BEST MODE FOR CARRYING OUT THE INVENTION

Description will be given below with respect to preferable embodimentsof the method for culturing cells and cell culturing apparatus accordingto the present invention by referring to the accompanying drawings.

First Embodiment

First, referring to FIG. 1 through FIG. 4, a first embodiment of thepresent invention will be described. FIG. 1 is a diagram illustratingthe cell culturing apparatus according to this embodiment. FIG. 2 is adiagram illustrating a driving device of the cell culturing apparatusaccording to this embodiment. FIG. 3 is a schematic side view of thecell culturing apparatus according to this embodiment. FIG. 4 is adiagram illustrating formation of an aggregate of cells anddisintegration of the aggregate of cells in the present invention.

[Cell Culturing Apparatus 10]

As shown in FIG. 1, a cell culturing apparatus 10 according to thisembodiment includes a culture container 11, a loading base 13, and anagitating member 14. A culture solution (culture medium) and cells areenclosed within a storage 11-1 of the culture container 11, and tubes 12are coupled to the storage 11-1.

The culture container 11 is a container that is made of a soft packingmaterial shaped in the form of a bag (bag type). The soft packingmaterial used as the material of the culture container 11 provides theculture container 11 with flexibility and plasticity. Examples of thesoft packing material include materials disclosed in JP2002-255277A(“FOOD PACKAGE USING SOFT PACKAGING FILM SHEET AND FOOD TAKING-OUTMETHOD”) and JP2004-323077A (“PRESSURIZEDLY SPOUTING BAG-SHAPEDCONTAINER”).

Also the culture container 11 has gas permeability required for cellculturing, and partially or entirely has transparency to permitconfirmation of the contents. Examples of the material of the culturecontainer satisfying the these conditions include polyolefins,ethylene-vinyl acetate copolymers, styrene elastomers, polyesterthermoplastic elastomers, silicone thermoplastic elastomers, andsilicone rubber.

The tubes 12 are used to inject a culture solution and cells into theculture container 11 from outside the culture container 11, and tocollect them to outside the culture container 11. Although each of thefour sides of the culture container 11 is sealed, at least two tubes 12are coupled to the culture container 11. One of the at least two tubes12 is used to inject cultured cells and a culture medium into theculture container 11 from outside the culture container 11, while theother tube is used to collect cultured cells and the culture medium outof the culture container 11. When three tubes 12 are attached as shownin FIG. 1, the third tube is used for sampling purposes, taking out thecultured cells and the culture medium as samples out of the culturecontainer 11.

The material of the tubes 12 may be selected appropriately in accordancewith the application environment. Examples include: silicone rubber;soft vinyl chloride resins; polybutadiene resins; ethylene-vinyl acetatecopolymers; chlorinated polyethylene resins; polyurethane thermoplasticelastomers; polyester thermoplastic elastomers; silicone thermoplasticelastomers; styrene elastomers such as SBS (styrene-butadiene-styrene),SIS (styrene-isoprene-styrene), SEBS(styrene-ethylene-butylene-styrene), and SEPS(styrene-ethylene-propylene-styrene); polyolefin resins; and fluorineresins.

The loading base 13 is a plane table with the culture container 11placed on the top face. On the top face of the culture container 11, theagitating member 14 is mounted.

At each of four corners of the top face of the loading base 13 where theculture container 11 is placed, a stopper 13-1 is disposed upright.Meanwhile, at each of four corners of the culture container 11, a hole11-2 is pierced through which the corresponding stopper 13-1 isinserted.

Inserting the stoppers 13-1 inserted through the holes 11-2 secures theculture container 11 on the top face of the loading base 13. This alsoprevents misalignment of the culture container 11 with the movement ofthe agitating member 14.

It should be noted that the stoppers will not be limited to the abovemembers. It is possible to use any type of stoppers insofar as thestoppers have a prevention mechanism of misalignment of the culturecontainer 11.

The agitating member 14 applies an external force onto the culturecontainer 11 to control cell aggregate formation and cell aggregatedisintegration with respect to cells disposed in the culture container11.

In the method for culturing cells according to this embodiment, as shownin FIG. 3, the agitating member 14 presses the culture container 11 to apredetermined pressing degree and moves at a predetermined speed inparallel to the loading base 13. This movement is repeated with apredetermined cycle. Examples of the agitating member 14 used include aroller.

In this manner, the agitating member 14 in this embodiment employsapplication an external force onto a culture container to ensure fineadjustment of agitation effected in the culture container 11. Thisensures appropriate agitation for cell aggregate formation and ensuresappropriate agitation for cell aggregate disintegration.

As shown in FIG. 1, a supporting stand 15 includes: upright bearingportions disposed at respective positions of both sides of the loadingbase 13 to rotatably support both ends of the agitating member 14; and aconnection member to couple the bearing portions to one another.

As shown in FIG. 2, the supporting stand 15 is movable upward anddownward by a rod type electric cylinder 17 (actuator for actuation inthe vertical direction) placed beneath the connection member. Thisensures fine adjustment, on a 0.1 mm basis, of the pressing degree ofthe agitating member 14 secured to the supporting stand 15 against theculture container 11.

Further, the rod type electric cylinder 17 is secured to a movingcarriage 16 on a slider type electric cylinder 21 (actuator foractuation in the horizontal direction) to permit movement in thehorizontal direction relative to the loading base 13. In order to adjustthe moving speed of the agitating member 14 secured to the supportingstand 15, the moving speed of the moving carriage 16 in the horizontaldirection is controlled.

The supporting stand 15, the moving carriage 16, the rod type electriccylinder 17, and the slider type electric cylinder 21 constitute adriving device of the cell culturing apparatus 10 according to thisembodiment.

Thus, with the cell culturing apparatus 10 according to this embodiment,the rod type electric cylinder 17 and the slider type electric cylinder21 are used to adjust the pressing degree of the agitating member 14against the culture container 11 and to adjust the moving speed of theagitating member 14. This ensures control of agitation of the culturesolution in the culture container 11 to optimize the size of aggregatesof the cells.

The operation control of the agitation member may be by other than theelectric actuators such as the rod type electric cylinder 17 and theslider type electric cylinder 21. It is also possible to use actuatorsutilizing air pressure, oil pressure, or an electromagnetic force, oruse motors and cams.

<Formation of Aggregate of Cells>

As shown in FIG. 4, in cell culturing, there is an appropriate size ofan aggregate of cells depending on the kind of cells used for culturing.

That is, cultured cells have such a characteristic that an individualcell has a low division rate, and it is when cells adhere to form acertain amount of aggregate that adequate division starts. In the usualstationary culturing, when the cell density is low at an early stage ofculturing, cells adhere by dispersion and gradually form aggregates,though at a comparatively low rate.

In view of this, conventional cell culturing uses, for example, a wellplate at an early stage of culturing to forcibly gather cells at oneplace of high density, where the cells adhere easily. Alternatively, acontainer of small capacity is used first and then a large container isused as the cells proliferate. Thus, the cells are cultured whilepreventing lowering of the cell density.

In contrast, in this embodiment, agitation causes the cells floating onthe bottom of the culture container 11 to move actively to increase theprobability of cell adhesion, ensuring earlier formation of aggregatesof appropriate size.

Consequently, according to the method for culturing cells employing thecell culturing apparatus 10 according to this embodiment, when the cellsare separated from each other at the time of start of cell culturing,the formation of aggregates of appropriate size is promoted by bringingthe cells into contact with each other. This improves cell proliferationefficiency.

Further, such cell aggregate formation control is not limited to anearly stage of culturing (at the time of seeding). Other suitableexamples include the case where the aggregates collapse and the cellsare separated from each other as a result of application of an excessexternal force to the culture container 11 during cell culturing. This,as a result, improves cell proliferation efficiency.

<Disintegration of Aggregate of Cells>

Meanwhile, if an aggregate of cells is excessively large, a lack ofoxygen and a lack of nutrients occur in the inner side of the aggregate,resulting in degraded cell proliferation efficiency.

In view of this, when an aggregate becomes large, it is preferable tocause a strong flow (turbulent flow) in the culture solution todisintegrate the aggregates.

According to the method for culturing cells employing the cell culturingapparatus 10 according to this embodiment, when an aggregate becomeslarge, the pressing degree and speed of the agitating member 14 isadjusted to control agitation to cause a strong flow in the culturesolution, thereby disintegrating the aggregate into an appropriate size.

As described above, with the cell culturing apparatus 10 according tothis embodiment and the method for culturing cells employing the cellculturing apparatus 10, the agitating member 14 presses the culturecontainer 11 to a predetermined pressing degree and moves at apredetermined speed in parallel to the loading base 13. This ensuresappropriate control of the strength of an external force applied ontothe culture container 11.

Consequently, the fine adjustment of the agitation in the culturecontainer 11 ensures appropriate agitation for cell aggregate formation,and also ensures appropriate agitation for cell aggregate disintegrationwhile ensuring that the cells are not separated from each other. Thisensures adjustment of the size of a cell aggregate to a size appropriatefor proliferation.

Second Embodiment

Next, referring to FIG. 5, a second embodiment of the present inventionwill be described. The figure is a diagram illustrating the cellculturing apparatus according to this embodiment.

This embodiment is different from the first embodiment in that the cellculturing apparatus 10 has the culture container 11 divided into aculture portion and an expansion portion by a partition member so as tomake the capacity of the culture medium adjustable to an appropriatesize in accordance with the proliferation of cells, with the cultureportion being agitatable by the agitating member 14 (agitating roller).Further, in this embodiment, both ends of the culture container 11 aresecured by clamp members 23. This embodiment is otherwise similar to thefirst embodiment.

That is, as shown in FIG. 5, in the cell culturing apparatus 10according to this embodiment, the culture container 11 is divided bypartition rollers (partition member) 22 to provide a culture portion inwhich a culture solution and cells are enclosed, and an expansionportion that expands the capacity of the culture portion with themovement of the partition rollers 22. The partition rollers 22 aredisposed in parallel to the agitating member (agitating roller) 14, andmovable in parallel to the loading base 13.

Use of the partition rollers 22 ensures a continuous change in thecapacity of the culture portion. That is, the partition rollers 22 movewith the proliferation of cells to increase the capacity of the cultureportion, thus maintaining the cell density within an appropriate range.

While in the example shown in the above figure the partition isimplemented by vertically pinching the culture container 11 between twopartition rollers 22, this should not be construed in a limiting sense.For example, it is also possible to use a single partition roller 22 topress from upward the culture container 11 against the loading base 13,thus partitioning the culture container 11 into a culture portion and anexpansion portion.

WO2008/136371 and WO2008/136339, both filed by the applicant, describe atechnique to control the culture capacity using a partition member so asto improve culture efficiency.

According to the cell culturing apparatus 10 according to thisembodiment and the method for culturing cells employing the cellculturing apparatus 10, use of the partition rollers 22 ensures controlof the culture capacity at a level that ensures high cell proliferationefficiency. Use of the partition rollers 22 also ensures adjustment ofthe size of a cell aggregate in the culture portion to a size thatensures high cell proliferation efficiency.

This results in further improved cell proliferation efficiency.

Third Embodiment

Next, a third embodiment of the present invention will be described byreferring to FIG. 6. This figure is a diagram illustrating the cellculturing apparatus 10 according to this embodiment.

This embodiment is different from the first embodiment in the followingrespects. The cells in the culture container 11 are photographed.Whether the size of an aggregate is within a predetermined range isdetermined automatically. The size of aggregates is adjusted to anappropriate size based on the determination. This embodiment isotherwise similar to the first embodiment.

Specifically, as shown in FIG. 6, the cell culturing apparatus 10according to this embodiment includes a photographing device 30 and acontrol device 40, in addition to the configuration of the firstembodiment.

Upon receipt of instruction information of photographing from thecontrol device 40, the photographing device 30 photographs the cells inthe culture container 11, and transmits the obtained image to thecontrol device 40. The instruction information of photographing for thephotographing device 30 may be transmitted automatically with apredetermined timing from the control device 40.

Examples of the photographing device 30 used include a CCD camerasecured to a lens barrel of a phase contrast microscope.

The control device 40 is an information processing device that controlsa driving device to move the agitating member 14 in the cell culturingapparatus 10 and controls the photographing device 30. As shown in FIG.6, the control device 40 includes a photographing device control unit41, an aggregate size determining unit 42, and a driving device controlunit 43.

The photographing device control unit 41 transmits the instructioninformation to cause the photographing device 30 to carry outphotographing with a predetermined timing, and receives the photographedimage from the photographing device 30.

When the photographing device control unit 41 receives an image, theaggregate size determining unit 42 determines whether the size of thecell aggregate in this image information is within a predeterminedrange. Examples of the predetermined range include 100 μm to 600 μm. Thesize of the aggregate may be the average value of photographedaggregates.

As a result of the determination, when the size of the cell aggregate isbelow or above the predetermined range, the determination result isoutput to the driving device control unit 43.

The driving device control unit 43 determines the pressing degree,moving speed, and moving cycle of the agitating member 14 based on thedetermination result input from the aggregate size determining unit 42,and controls the rod type electric cylinder 17 and the slider typeelectric cylinder 21 based on these driving conditions.

Thus, when the size of the cell aggregate is below the predeterminedrange, the cell aggregate in the culture container 11 is increased to anappropriate size, while when the size of the cell aggregate is above thepredetermined range, the cell aggregate is disintegrated into anappropriate size. This ensures adjustment of the size of the cellaggregate to an optimal size for proliferation.

In order to carry out this processing, the control device 40 or thedriving device control unit 43 preferably has a table that memorizesvarious aggregate sizes in relation to data of appropriate pressingdegree, moving speed, and moving cycle of the agitating member 14.

As described above, with the cell culturing apparatus 10 according tothis embodiment and the method for culturing cells employing the cellculturing apparatus 10, the size of an aggregate in the culturecontainer 11 is adjusted automatically. This improves cell proliferationefficiency stably.

Next, referring to FIG. 7, description will be given with regard to aprinciple of the method for counting counted targets disposed in acontainer according to an embodiment of the present invention. Thefigure shows how the cells in the culture container are directlyobserved through a microscope and the number of the cells is counted.FIG. 7(A) shows conventional observation, which does not involveadjustment of the thickness of the container. Use of a culture solutionhaving a specific gravity smaller than that of the cells causes thecells to sink to the bottom of the container, which is a suitable statefor observation through a microscope. It is also possible to use aculture solution having a large specific gravity, in which case thecells are gathered at an upper portion of the container, where the cellsare observed.

In the case of FIG. 7(A), as the number of cells in the containerincreases, the cells gradually overlap with each other when the cellsare suspended and made stationary. Accordingly, this method does notprovide accurate counting of the number of cells in large-scale cellculturing.

In view of this, in this embodiment of the present invention, when thenumber of the cells is excessively high for accurate correcting, thethickness of the container is reduced as shown in FIG. 7(B) to reducethe number of the cells in an observation region (measurement targetregion), thus adjusting the number of the cells to a number suitable formeasurement.

Thus, even in the case of large-scale cell culturing using a culturecontainer, the direct observation of cells in the culture containerensures counting of the number of cells without disassembling of theculture system.

Further, when the number of cells is excessively small in the culturecontainer and thus it is difficult to prospect the density throughoutthe culture container, increasing the thickness of the containerincreases the number of the cells in the observation region, thusadjusting the number of the cells to a number suitable for measurement.

Fourth Embodiment

Next, referring to FIG. 8, description will be made with respect to amethod for counting counted targets disposed in a container and acounting apparatus according to a fourth embodiment of the presentinvention. The figure is a diagram illustrating the counting apparatusaccording to this embodiment. As shown in the figure, a countingapparatus 50 according to this embodiment includes a thickness adjustingmember 51, a loading base 13, photographing means 52, a driving device53, a driving device 54, and an illumination source 55. With culturecontainer 11 disposed on the loading base 13, the number of culturedcells in the culture container 11 is counted.

The thickness adjusting member 51 adjusts the thickness of the culturecontainer 11 on the loading base 13. In the example shown in FIG. 8, thethickness adjusting member 51 includes a pressing plate having a flatportion used to press the culture container 11, and presses from upwarda part of the culture container 11, which is made of a soft packingmaterial, from the upper side to reduce the thickness of the culturecontainer 11.

At least a part of the thickness adjusting member 51 located above themeasurement target region of the culture container 11 is made of atransparent material. This ensures observation from downward by thephotographing means 52, which includes a microscope and a CCD camera,with illumination from upward by the illumination source 55.

The thickness adjusting member 51 is not limited to the pressing plateto press the culture container 11 with the flat portion as shown in FIG.8. For example, the thickness adjusting member 51 may include rollers ora stretching member.

For example, rollers may move while vertically nipping the culturecontainer 11 from both sides or one side to reduce the horizontal areaof the culture container 11, thus increasing the thickness of theculture container 11. In contrast, increasing the horizontal area of theculture container 11 reduces the thickness of the culture container 11.When a stretching member is used to stretch the culture container 11 inthe horizontal direction, the thickness of the culture container 11 isalso reduced.

The loading base 13 is a flat base on which the culture container 11 isplaced, and constitutes a placing device for counting, together with thethickness adjust member 51.

A part of the loading base 13 positioned beneath the measurement targetregion is made of a transparent member such as a glass plate 56 topermit observation of the culture container 11 from downward by thephotographing means 52.

The driving device 53 moves the thickness adjusting member 51 upward anddownward via ball screws as shown in FIG. 8. This permits the thicknessadjusting member 51 to press the culture container 11 on the loadingbase 13 and to adjust the thickness of the culture container 11.

Examples of the driving device 53 include a rod type electric cylinder(actuator for actuation in the vertical direction). This ensures thatthe thickness of the culture container 11 is finely adjusted, on a 0.01mm basis.

The driving device 54 moves the photographing means 52 in the horizontaldirection relative to the loading base 13 by ball screws as shown inFIG. 8. The driving device 54 keeps the photographing means 52 arrangedoutside the loading base 13 except during photographing of cells in theculture container 11, and at the time of photographing, moves thephotographing means 52 to a position beneath the measurement targetregion of the culture container 11.

The driving device 53 and the driving device 54 may be other thanelectric actuators. It is also possible to use actuators utilizing airpressure, oil pressure, or an electromagnetic force, or use motors andcams.

The illumination source 55 illuminates the measurement target region inthe culture container 11 through the thickness adjusting member 51, andprovides brightness required for photographing of cells by thephotographing means 52. In this respect, the amount of transmitted lightvaries depending on the adjusted thickness of the culture solution,which may cause difference in contrast among photographed images. Inview of this, it is preferable to adjust the amount of light inaccordance with the thickness of the culture solution.

The culture container 11 may be similar to that in the first embodiment.

Next, referring to FIG. 9 and FIG. 10, description will be made withrespect to an example of the adjustment of the thickness of the culturecontainer 11 by the counting apparatus according to this embodiment.FIG. 9 shows a case where the thickness of the container is reduced, andFIG. 10 shows a case where the thickness of the container is increased.

When the thickness of the culture container 11 is reduced, theadjustment of the thickness of the container may be other than theexample shown in FIG. 8. For example, as shown in (a) of FIG. 9, thethickness adjusting member 51 may press the culture container 11 fromdownward. In this respect, it is preferable to fix the position of theupper face of the culture container 11 with a glass plate or the like,an illustration of which is omitted.

When the culture container 11 is made of a horizontally stretchablematerial, the thickness of the culture container 11 may be reduced bypressing the culture container 11 using a thickness adjusting member 51capable of covering the entire surface of the culture container 11 asshown in (b) of FIG. 9. As shown in (c) of FIG. 9, it is also possibleto reduce the thickness of the culture container 11 by stretching theculture container 11 using a thickness adjusting member 51. In thiscase, the culture container 11 may be stretched with one edge of theculture container 11 fixed and the other edge opposite in the horizondirection stretched using a thickness adjusting member 51 that includesa stretching member. Alternatively, both ends of the culture container11 may be stretched in the horizon direction using two thicknessadjusting members 51. Examples of the material of the flexible culturecontainer 11 include silicone rubbers.

As shown in (d) of FIG. 9, it is also possible to use rollers and thelike as thickness adjusting members 51, in which case the rollers moveto increase the horizontal area of the culture container 11, thusreducing the thickness of the culture container 11.

When the thickness of the container is increased, for example, as shownin (e) of FIG. 10, the thickness adjusting member 51 may press a part ofthe top face of the culture container 11. This increases the thicknessof a part of the culture container 11 other than the pressed part.

As shown in (f) of FIG. 10, it is also possible to use rollers and thelike as thickness adjusting members 51, in which case the rollers moveto reduce the horizontal area of the culture container 11, thusincreasing the thickness of the culture container 11.

Next, a method for counting counted targets in the container accordingto this embodiment will be described in detail by referring to FIG. 8.

First, it is preferable to agitate the culture solution disposed in theculture container 11 prior to the step of counting the number of thecells disposed in the culture container 11. It is noted that theagitating means for a culture solution will be described in detail inthe fifth embodiment.

Next, the driving device 54 moves the photographing means 52 to beneaththe measurement target region of the culture container 11.

Next, the driving device 53 moves the thickness adjusting member 51downward by to adjust the thickness of the culture container 11 to apredetermined thickness.

In this respect, the predetermined thickness may be determined onvarious values depending on the kind of the cells, the size of theculture container, the area of the measurement target region, and theperiod for culturing.

Next, the measurement target region is illuminated by the illuminationsource 55, and photographed by the photographing means 52. Then, thenumber of cells in the photographed image is counted. In this respect,the photographed image is transmitted from the photographing means 52 tocounting means and the number of the cells are automatically counted bythe counting means. The counting means may be a known cell countinganalyzing device and cell counting apparatus.

In this embodiment, the culture solution has a specific gravity smallerthan that of cultured cells, and hence the cultured cells areprecipitated on the bottom of the culture container 11. Thephotographing means focuses on the precipitated cells to photograph thecells. When, however, the cells have a large density and overlap witheach other in the measurement target region, the number of cells may notbe counted accurately.

In view of this, in the method for counting counted targets disposed ina container according to this embodiment, when cells overlap with eachother in the measurement target region as described above, the thicknessadjusting member 51 moves to reduce the thickness of the culturecontainer 11 and thus to reduce the number of cells in the measurementtarget region. This ensures a countable number of cells.

The maximum number of cells that can be observed without celloverlapping in the measurement target region may be assumedapproximately at less than a number obtained by dividing the area of themeasurement target region by the average horizontal area of the culturedcells. In view of this, when as a result of counting of the number ofcells, the counted number of cells is equal to or more than the maximumnumber of cells, it is preferable to reduce the thickness of the culturecontainer 11 and then carry out the counting again. It is furtherpossible to use other values in determining whether to adjust thethickness of the culture container 11, examples of the values include anaccurately predicted number of densely arranged cells in the measurementtarget region. The predicted number may be used as the maximum number ofcells.

Furthermore, when as a result of counting of the number of cells, thecounted number of the cells is less than a predetermined value, theaccuracy of the resultant cell density is low. In view of this, it ispreferable to increase the thickness of the culture container 11 andthen carry out the counting again. The sixth embodiment describes indetail a counting apparatus used for the above-described increasing ofthe thickness of the culture container 11.

The number of the cells in the measurement target region is counted inthis manner, and then the number of cells is divided by the volume ofthe measurement target region, thus calculating the cell density.Further, the obtained cell density is multiplied by the volume of theculture container 11, thus calculating the number of cells disposedthroughout the culture container 11.

The counting means is able to automatically calculate the cell densityand the number of cells disposed throughout the culture container 11.

Thus, according to this embodiment, even when the number of cells in theculture container 11 is excessively large for accurate counting of thenumber of cells by direct observation of the culture container 11,reducing the thickness of the culture container 11 ensures counting ofthe number of cells. This ensures calculation of the density of thecells in the culture container 11.

Fifth Embodiment

Next, referring to FIG. 11 and FIG. 2, description will given withregard to a counting method and a counting apparatus to count countedtargets disposed in a container according to the fifth embodiment of thepresent invention. FIG. 11 is a diagram illustrating the countingapparatus according to this embodiment. FIG. 2 is a diagram illustratinga driving device (for an agitating member) of the cell culturingapparatus according to the first embodiment. In this embodiment, asimilar apparatus may be used.

The counting apparatus according to this embodiment includes anagitating member in addition to the configuration of the countingapparatus according to the fourth embodiment. In this embodiment,agitating the culture solution disposed in the culture container 11using the agitating member disperses the culture solution to make thecells easily observable. This embodiment may be otherwise similar to thefourth embodiment.

The agitating member 14 moves while pressing the culture container 11 toagitate the culture solution in the culture container 11 and dispersethe cultured cells in the culture solution. Examples of the agitatingmember 14 include a roller as shown in FIG. 11.

In the example shown in this figure, the agitating member 14 presses theculture container 11 to a predetermined pressing degree, and at the sametime, the agitating member 14 moves at a predetermined speed in parallelto the loading base 13 repeatedly with a predetermined cycle, thusagitating the culture solution.

As shown in FIG. 11, a supporting stand 15 includes: upright bearingportions disposed at respective positions of both sides of the loadingbase 13 to rotatably support both ends of the agitating member 14; and aconnection member to couple the bearing portions to one another.

As shown in FIG. 2, the supporting stand 15 is movable upward anddownward by a rod type electric cylinder 17 (actuator for actuation inthe vertical direction) placed beneath the connection member. Thisensures fine adjustment, on a 0.1 mm basis, of the pressing degree ofthe agitating member 14 secured to the supporting stand 15 against theculture container 11.

Further, the rod type electric cylinder 17 is secured to a movingcarriage 16 on a slider type electric cylinder 21 (actuator foractuation in the horizontal direction) to permit movement in thehorizontal direction relative to the loading base 13. In order to adjustthe moving speed of the agitating member 14 secured to the supportingstand 15, the moving speed of the moving carriage 16 in the horizontaldirection is controlled.

The operation control of the agitation member 14 may be by other thanthe electric actuators such as the rod type electric cylinder 17 and theslider type electric cylinder 21. It is also possible to use actuatorsutilizing air pressure, oil pressure, or an electromagnetic force, oruse motors and cams.

With the counting method and counting apparatus to count counted targetsdisposed in a container according to this embodiment, prior to countingthe number of cells in the culture container 11, the agitating member 14makes reciprocating movement in the horizontal direction for apredetermined period of time with the agitating member 14 pressing theculture container 11 to a predetermined pressing degree.

This ensures that the culture solution in the culture container 11 isagitated to disperse the cultured cells disposed in the culturecontainer 11 in a manner that facilitates counting of the number of thecultured cells.

Sixth Embodiment

Next, referring to FIG. 12 through FIG. 16, description will given withregard to a counting method and a counting apparatus to count countedtargets disposed in a container according to the sixth embodiment of thepresent invention. These figures are schematic diagrams showing a basicposition (FIG. 12) of the counting apparatus in the method for countingcounted targets disposed in a container according to this embodiment, astate of agitation (FIG. 13), a state of thickness regulation andprecipitation waiting (when the number of cells to be counted is desiredto be reduced: FIG. 14), a state of microscope observation (FIG. 15),and a state of thickness regulation and precipitation waiting (when thenumber of cells to be counted is desired to be increased: FIG. 16).

The counting apparatus according to this embodiment includes a thicknessadjusting member to increase the thickness of the culture container 11in addition to the configuration of the counting apparatus according tothe fifth embodiment. This embodiment may be otherwise similar to thefifth embodiment.

Description will be given with regard to the operation procedure of thecounting apparatus in the method for counting counted targets disposedin a container according to this embodiment of the present invention,along with the operations of increasing and reducing the thickness ofthe culture container 11.

(1) Basic Positions

First, referring to FIG. 12, the basic positions of the structuralelements of the counting apparatus according to this embodiment will bedescribed.

In this figure, the loading base 13 includes an observation hole forobservation via a microscope, and a glass plate 56 constituting a partof the top face of the loading base 13 is fit in the upper part of theobservation hole.

The culture container 11 is disposed on the loading base 13, and athickness adjusting member 51-1 (pressing plate) is disposed above theobservation hole of the loading base 13. The thickness adjusting member51-1 moves downward to press the culture container 11, thus reducing thethickness of the culture container 11.

A thickness adjusting member 51-2 (roller) is disposed at one edge ofthe culture container 11. The thickness adjusting member 51-2 moves toreduce the horizontal area of the culture container 11, thus increasingthe thickness of the culture container 11.

The agitating member 14 (roller) is disposed above the culture container11. The agitating member 14 moves downward, and then the agitatingmember 14 moves in the horizontal direction while pressing the culturecontainer 11 to agitate the culture solution disposed in the culturecontainer 11.

Regarding the basic positions, a microscope 52-1 and a CCD camera 52-2that constitute the photographing means 52 for photographing cells, andthe illumination source 55 are arranged outside the loading base 13.

(2) Agitating State

Next, referring to FIG. 13, description will be made with respect to theprocedure for agitating the culture solution in the culture container 11prior to counting the number of counted targets in a container.

First, the agitating member 14 moves downward from its basic positionshown in FIG. 12, and presses the culture container 11 to apredetermined pressing degree. Next, the agitating member 14 repeats apredetermined cycle of movement in parallel to the loading base 13 at apredetermined speed.

This ensures agitation of the culture solution disposed in the culturecontainer 11 and equalization of the cells in the culture solution.

(3) Thickness-Regulating and Precipitation-Waiting State (ThicknessReduction)

Next, referring to FIG. 14, description will be made with respect to theprocedure for reducing the thickness of the culture container 11 so asto reduce the number of cells to be counted.

First, the agitating member 14 moves upward to return to its basicposition shown in FIG. 12, and then, the thickness adjusting member 51-1moves downward to reduce the thickness of the culture container. In thisrespect, the thickness adjusting member 51-1 presses a part of theculture container 11 including a region above the observation hole ofthe loading base 13, thus adjusting the thickness of the measurementtarget region of the culture container 11 to a predetermined size. Inthis respect, under the culturing environment, as the thickness of theculture container 11 reduces, the number of cells to be counted reduces.

After the thickness of the culture container 11 is reduced, the culturecontainer 11 is kept stationary until the cells in the culture container11 precipitate.

(4) Microscope Observation State

Next, referring to FIG. 15, description will be made with respect to theprocedure for counting the number of cells.

After the cells in the culture container 11 precipitate with thethickness of the culture container 11 reduced, as shown in FIG. 15, thephotographing means, which includes the microscope 52-1 and the CCDcamera 52-2, and the illumination source 55 move in the horizontaldirection from their respective basic positions shown in FIG. 12 so thatthe photographing means 52 is disposed immediately beneath theobservation hole and the illumination source 55 is disposed above theobservation hole. Then, the cells that precipitate in the culturecontainer 11 are photographed with the CCD camera 52-2.

The image captured in this manner is input to a counting apparatus, notshown, and the number of the cells in the image is counted by thecounting apparatus. The density of the cells in the culture container 11is calculated by dividing the obtained number of the cells by the volumeof the measurement target region (the region of the culture container 11observed by the CCD camera 52-2).

Thus, adjusting the thickness of the culture container 11 ensuresaccurate cell counting in the case where the cells in the culturecontainer 11 proliferate enough to make their accurate countingdifficult due to overlapping if the cells precipitate with the thicknessof the culture container 11 remaining unchanged. This ensurescalculation of the density of cells in the culture container 11 withoutdisassembly of the culture system.

(5) Thickness-Regulating and Precipitation-Waiting State (ThicknessIncrement)

In an early stage of culturing, for example, the number of the cells issmall. Although counting may be possible, the accuracy of the obtaineddensity of the cells can turn out to be low.

In view of this, when the number of the cells is small, the thickness ofthe culture container 11 is increased as shown in FIG. 16 so as toincrease the number of the cells in the measurement target region, priorto the microscope observation described in (4).

Specifically, after the agitation in (2), the agitating member 14 movesupward to return to its basic position shown in FIG. 12. Then, thethickness adjusting member 51-2 including a roller moves in thedirection of the measurement target region to press the culturecontainer 11, thus increasing the thickness of the culture container 11.In this respect, in order to make the thickness of the culture container11 uniform in the measurement target region, the thickness adjustingmember 51-1 moves to a position of contact with the top face of theculture container 11 on the measurement target region. Then, the culturecontainer 11 is kept stationary until the cells in the culture container11 precipitate.

This increases the number of the cells in the measurement target region,and ensures more accurate calculation of the density of the cells whenthe number of the cells in the culture container 11 is small.

A specific cell number for the smallness of the number of the cells inthe culture container 11 may be determined based on the kind of thecells and the area of the measurement target region. Exemplary numbersinclude 0 and less than 10.

After the thickness of the culture container 11 is adjusted inaccordance with the procedure of (1) through (5) described above, thethickness of the culture container 11 is adjusted again in accordancewith the procedure of (1) through (5) and the number of cells is countedif any of the following occurs: overlapping is observed among the cells;no cell is observed; and approximately no cell is observed in themeasurement target region.

Thus, with this embodiment, increasing the thickness of the culturecontainer 11 ensures accurate calculation of the density of the cells inthe culture container 11 when the number of the cells is small and anaccurate cell density may not be obtained by observing the culturecontainer 11 with the culture container 11 remaining unchanged.

EXAMPLE

Next, description will be made with respect to examples in which cellswere cultured by the cell culturing apparatus 10 according to the firstembodiment. First, referring to FIG. 17 and FIG. 18, various types ofculture conditions and the degree of agitation in the respective typesare described. FIG. 17 is a diagram illustrating kinds of conditions ofagitation by the cell culturing apparatus 10 according to thisembodiment, and FIG. 18 is a diagram showing cell states as a result ofagitation under the agitation conditions.

As shown in FIG. 17, with the culturing method according to theembodiment of the present invention, the agitating member 14 presses theculture container 11 from upward, and then the agitating member 14 movesin parallel to the loading base 13, thus agitating the culture medium inthe culture container 11.

The pressing degree of the agitating member 14 pressing the culturecontainer 11 may be set at various values. For example, as shown in thefigure, when the thickness of the culture container 11 is 10.5 mm, thepressing degree may be set at 2 mm, 4 mm, 6 mm, and 8 mm.

Further, the moving speed of the agitating member 14 is adjusted inaccordance with the respective pressing degrees. This is for the purposeof controlling the agitation of the culture solution disposed in theculture container 11 in order to obtain appropriate agitation for cellaggregate formation and appropriate agitation for cell aggregatedisintegration.

Examples of the moving speed of the agitating member 14 include 2.5mm/s, 12.5 mm/s, and 50 mm/s, as shown in the figure.

While there is no specific limitation to the diameter of the agitatingmember 14, the diameter is preferably 0.5 time to 3.0 times thethickness of the culture container 11 for appropriate control of theagitation.

FIG. 18 shows how much the culture solution in the culture container 11was agitated under the above-described agitation conditions.

As shown in the figure, all the cells in the culture container 11 keptdescended at a pressing degree of 2 mm and at a moving speed of 2.5 mm/sor 12.5 mm/s; and at a pressing degree of 4 mm and at a moving speed of2.5 mm/s.

In the present specification, such agitation is referred to as “weakagitation”. Agitation in accordance with “weak agitation” promotes cellaggregate formation.

Meanwhile, the culture solution in the culture container 11 was agitatedto some extent, but most of the cells kept descended at a pressingdegree of 2 mm and at a moving speed of 50 mm/s; at a pressing degree of4 mm and at a moving speed of 12.5 mm/s or 50 mm/s; at a pressing degreeof 6 mm and at a moving speed of 2.5 mm/s or 12.5 mm/s; and at apressing degree of 8 mm and at a moving speed of 2.5 mm/s, as shown inFIG. 18.

Further, the culture solution in the culture container 11 was agitatedto some extent, and most of the cells kept afloat at a pressing degreeof 6 mm and at a moving speed of 50 mm/s; and at a pressing degree of 8mm and at a moving speed of 12.5 mm/s.

In the present specification, the agitating condition in the vicinity ofthe boundary between the state in which most of the cells keep descendedand the state in which most of the cells keep afloat is referred to as“medium agitation”. Agitation in accordance with “medium agitation”ensures adjustment of a cell aggregate to an appropriate size withoutexcessive disintegration of the cell aggregate.

Meanwhile, the culture solution in the culture container 11 was agitatedintensely and all the cells kept afloat at a pressing degree of 8 mm andat a moving speed of 50 mm/s in FIG. 18. In the present specification,the agitation that makes all the cells afloat is referred to as “strongagitation”. Such “strong agitation” disintegrates a cell aggregate intoindividual cells. Repeating the “strong agitation” turns the cells intosuspension state. However, if cells are separated from each other,proliferation efficiency degrades.

In the agitation in the conventional cell culturing, a culture containeris agitated intensely, which corresponds to “strong agitation”. This isproblematic in that the agitation causes degradation of proliferationefficiency. The embodiments of the present invention solve this problem.

Next, description will be made with respect to examples in which thethickness of the culture container 11 was adjusted and then the numberof cells was counted by the counting method and counting apparatus tocount counted targets in a container according to the embodiment of thepresent invention. Description will be also made with respect to acomparative example in which the number of cells was counted withoutadjusting the thickness of the culture container 11.

Example 1

As the culture container 11, a bag made of LDPE (linear low-densitypolyethylene) with a film thickness of 0.15 mm was used. As shown inFIG. 19, the culture container 11 was partitioned with a partitionmember (rubber roller) to form a culture portion serving as a culturingregion with a longer side of 250 mm and a shorter side of 210 mm. Aculture solution of 640 ml was put into the culture portion. In thisrespect, the thickness of the culture container 11 placed in flatorientation was approximately 16 mm, though the top face of the culturecontainer 11 was not a perfectly horizontal surface. The horizontalsurface of the observation region (measurement target region) was, interms of size, a 0.5 mm by 0.5 mm square.

As the culture solution, AlyS5050N-0 culture medium available from CellScience & Technology Institute, Inc. was used. The cultured cells usedwere JurkatE6.1 strains of human leukemia T lymphoma proliferated to arequired amount in a cell culturing dish.

As the agitating member 14, a roller of 12 mm in diameter was used.Agitation was carried out under the agitation conditions of a pressingdegree of 13 mm, a speed of 50 mm/s, and 10 times of reciprocation.

Immediately after the agitation, a thickness adjusting member made of anacrylic board with a width of 50 mm, a thickness of 3 mm, and a lengthlonger than the longer side of the culture container 11 moved downwardonto the bag to adjust the thickness of the bag to 3.1 mm. Then, the bagwas kept stationary for 12 minutes, and then the observation region wasphotographed, followed by counting of the number of the cells. Then, thecell density was calculated based on the obtained cell number and thevolume of the observation region. FIG. 20 shows a photographed image,and FIG. 21 shows the number of the cells in the observation region, thecell density, an actual measurement density, and an actual measurementratio.

The actual measurement density was obtained such that the number of thecells in the culture solution collected from the bag was measured usinga counter board (OneCell counter (available from OneCell Corporation))according to a conventional method, and the measured number was dividedby the volume of the observation region.

Comparative Example 1

The agitation was carried out under the same agitating conditions withthe same culture container 11 and the culture solution as those inexample 1.

Next, after the bag was kept stationary for 60 minutes without beingadjusted in thickness, the observation region was photographed. Sincethe upper face of the bag was not pressed, its upper surface was in theform of a convexo-concave wave. The thickness of the bag wasapproximately 16 mm.

In this case, overlapping was observed among the cells, which made itimpossible to count the number of the cells and thus impossible tocalculate the density. The results are shown in FIG. 20 and FIG. 21.

Example 2

Example 2 was carried out in the same manner as in example 1 except thatthe culture solution used had a different cell density from the celldensity in example 1; the thickness of the bag was 11.0 mm in thecounting of the number of the cells; and the period of time for keepingthe bag stationary after the adjustment of the thickness was 45 minutes.The kind of the culture solution and the kind of the cultured cells werethe same as those in example 1. The number of the cells used in thisexample was smaller than that in example 1. The results are shown inFIG. 22 and FIG. 23.

Example 3

Example 3 was carried out in the same manner as in example 2 except thatin the measurement of the number of the cells, the thickness of the bagwas 7.0 mm, and the period of time for stationary keeping thereafter was30 minutes. The results are shown in FIG. 22 and FIG. 23.

Example 4

Example 4 was carried out in the same manner as in example 2 except thatin the measurement of the number of the cells, the thickness of the bagwas 4.0 mm, and the period of time for stationary keeping thereafter was12 minutes. The results are shown in FIG. 22 and FIG. 23.

Example 5

Example 5 was carried out in the same manner as in example 2 except thatin the measurement of the number of the cells, the thickness of the bagwas 3.1 mm, and the period of time for stationary keeping thereafter was12 minutes. The results are shown in FIG. 22 and FIG. 23.

As shown in FIG. 20 and FIG. 21, in comparative example 1, in which thethickness of the bag was not adjusted, the cells overlapped with eachother and the counting of the cell number was inaccurate. Examples ofthe impossibility of counting include the case where the result of thenumber counting is equal to or more than a predetermined value (themaximum number of cells viewable in the observation region), in additionto the case where the number cannot be actually calculated.

In contrast, in example 1, reducing the thickness of the bag ensuredmeasurement of the number of the cells, and ensured calculation of thedensity of the cells disposed in the culture container 11.

Also FIG. 22 and FIG. 23 show that in Examples 2 through 5, as thethickness of the bag reduces, the measured number of the cells lowers.It is also shown that the calculated cell densities are notsignificantly different from the corresponding actual measurementdensities.

Thus, it has been found that when the number of the cells is excessivelylarge for number counting, as the density of the cells is larger,reducing the thickness of the bag to a larger degree ensures measurementof the cells.

These experimental results clearly show that with the method forcounting counted targets disposed in a container using the countingapparatus according to the embodiment of the present invention, thenumber of cells in a culture container is measured and the density ofcells is calculated without disassembly of the culture system andirrespective of the density of cells.

The present invention is not limited to the above embodiments, and itwill be appreciated that various changes and modifications may be madewithin the scope of the present invention.

As examples of changes and modifications, the culture container 11 maybe rounded to eliminate corners, and the agitating member 14 may haveother than a columnar shape and may have the cross section shown in FIG.17 formed into various cross sections such as a star, so as to obtainvarious agitation effects.

While in the above-described embodiments and examples the cultured cellsare the measurement target, this should not be construed in a limitingsense. For example, the measurement target may be other organisms suchas plankton and inorganic substances. Examples of the “liquid” in theculture container include semi-liquids in addition to liquids such as aculture solution. It is also possible to use a liquid having a specificgravity larger than the specific gravity of the cultured cells as aculture solution in the culture container 11. This makes the culturedcells located at the upper portion in the culture container 11, and thenumber counting may be with respect to such cultured cells. Further, inthe above-described embodiments and examples, it is also possible toobserve the growth state of cells, as well as counting the number of thecells.

INDUSTRIAL APPLICABILITY

The present invention finds applications in fields that involveculturing of a large quantity of cells, such as biomedicine,regenerative medical therapy, and immunotherapy.

1. A method for culturing cells using a culture container, the methodcomprising applying an external force to the culture container to carryout at least one of cell aggregate formation control and cell aggregatedisintegration control with respect to cells disposed in a culturesolution in the culture container.
 2. The method for culturing cellsaccording to claim 1, wherein the applying step of the external force tothe culture container comprises: pressing an agitating member onto a topsurface of the culture container placed in flat orientation to apredetermined pressing degree; and moving the agitating member in ahorizontal direction at a predetermined speed, whereby the culturesolution in the culture container is agitated by the applying step ofthe external force to the culture container, thus controlling at leastone of cell aggregate formation and cell aggregate disintegration withrespect to the cells in the culture container.
 3. The method forculturing cells according to claim 1, wherein the cell aggregateformation is carried out with respect to cells seeded in the culturecontainer at a start of culturing.
 4. The method for culturing cellsaccording to claim 1, wherein the cell aggregate disintegration controlsan aggregate of the cells to a size within a predetermined range.
 5. Acell culturing apparatus comprising: a loading base on which a cellcontainer is placed, the cell container enclosing a culture solutioncomprising a culture medium and cells; and an agitating memberconfigured to press the culture container to a predetermined pressingdegree and movable at a predetermined speed in a horizontal direction,wherein the agitating member is configured to move to agitate theculture solution in the culture container so as to control at least oneof cell aggregate formation and cell aggregate disintegration withrespect to the cells in the culture container.
 6. The cell culturingapparatus according to claim 5, wherein the agitating member has aroller shape.
 7. The cell culturing apparatus according to claim 5,further comprising: a photographing device configured to photograph thecells in the culture container; determination means for receiving animage photographed by the photographing device and for determiningwhether an aggregate of the cells has a size within a predeterminedrange; and a driving device configured to, following a determination ofthe determination means, move the agitating member in the horizontaldirection at the predetermined speed with the predetermined pressingdegree so as to carry out the cell aggregate formation when the size ofthe aggregate of the cells is below the predetermined range, andconfigured to move the agitating member in the horizontal direction atthe predetermined speed with the predetermined pressing degree so as tocarry out the cell aggregate disintegration when the size of theaggregate of the cells is above the predetermined range.
 8. The cellculturing apparatus according to claim 5, further comprising a partitionmember configured to divide the culture container into two or morecompartments comprising a culture portion and an expansion portion, andconfigured to expand a capacity of the culture portion in accordancewith an increase in a number of the cells in the culture portion, theagitating member being configured to agitate the culture medium in theculture portion.
 9. A method for counting counted targets disposed in aliquid enclosed in a container, the method comprising: adjusting athickness of at least a part of the container; setting at least a partof the adjusted part as a measurement target region; and counting anumber of counted targets in the measurement target region.
 10. Themethod for counting counted targets disposed in a container according toclaim 9, further comprising, prior to adjusting the thickness of atleast a part of the container, agitating the liquid in the container toequalize the counted targets in the liquid.
 11. The method for countingcounted targets disposed in a container according to claim 9, furthercomprising, based on the counted number of the counted targets,calculating a density of the counted targets in the liquid, and/orcalculating a number of counted targets throughout the liquid.
 12. Themethod for counting counted targets disposed in a container according toclaim 9, the counting step of the number of the counted targetscomprises counting the number of the counted targets in a photographedimage of the measurement target region.
 13. The method for countingcounted targets disposed in a container according to claim 9, furthercomprising reducing a thickness of at least a part of the container whenthe number of the counted targets in the measurement target region isequal to or more than a predetermine value, and then counting the numberof counted targets.
 14. The method for counted targets in a containeraccording to claim 13, wherein the reducing step of the thickness of atleast a part of the container comprises pressing or stretching thecontainer using a thickness adjusting member.
 15. The method forcounting counted targets disposed in a container according to claim 9,further comprising increasing a thickness of at least a part of thecontainer when the number of the counted targets in the measurementtarget region is less than a predetermine value, and then counting thenumber of counted targets.
 16. The method for counting counted targetsdisposed in a container according to claim 15, wherein the increasingstep of the thickness of at least a part of the container comprisespressing the container using a thickness adjusting member.
 17. Themethod for counting counted targets disposed in a container according toclaim 9, wherein the container comprises a culture container, and thecounted targets comprise cells.
 18. A counting apparatus to countcounted targets disposed in a liquid enclosed in a container, thecounting apparatus comprising: a loading base on which the container isplaced; and an adjusting member configured to adjust at least a part ofthe container including a measurement target region to a predeterminedthickness.
 19. The counting apparatus according to claim 18, furthercomprising an agitating member configured to agitate the liquid in thecontainer before the adjusting member adjusts the thickness of thecontainer.
 20. The counting apparatus according to claim 18, furthercomprising: photographing means for photographing counted targetsdisposed in the container; counting means for counting a number of thecounted targets in a photographed image; and a driving device configuredto, when the number of the counted targets is outside a predeterminedrange following the counting by the counting means, drive the adjustingmember to adjust at least a part of the container to a predeterminedthickness so as to render the number of the counted targets in the imagewithin the predetermined range.